DOPAMINE TRANSPORTER--HUMAN & MOUSE GENES, DOPAMINERGIC DISORDERS & KNOCKOUT MICE

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The rewarding/reinforcing effects of cocaine are produced by blocking reuptake of dopamine by the dopamine transporter (DAT). The DAT gene is expressed in dopaminergic neurons of the ventral midbrain, and serves as the only currently-available marker expressed almost exclusively by these cells. During this FY, we have completed sequencing of the human DAT gene of fifteen exons and fourteen introns with the exception of a highly polymorphic region whose position has not been accurately determined. We have characterized additional intron specific sequences and found one to be polymorphic in intron 8. We have identified new alleles at the genetic marker D5S678, and allele frequencies at this site in caucasian and african-americans. The wide distribution of mRNA expression of the new gene found by sequence match to various EST clones was determined. The 5'-end of this gene has not been mapped successfully and may be in close enough proximity to the DAT VNTR to be a candidate for the recent associations of the VNTR to cocaine-induced paranoia and attention deficit/ hyperactivity disorder. We have confirmed in 297 individuals that linkage disequilibrium values are very low for the VNTR marker in exon 15 and the TaqI RFLP in intron 3, which suggests that a significant amount of recombination has occurred within this gene since the origins of humans. A region within intron 14 of the DAT gene has been successfully cloned and sequenced. This region was not present in several genomic libraries and even proved difficult to clone from PCR fragments. The probable cause of the difficulty was evident from the presence of a homopolymeric run of 17 adenines flanked by a direct repeat of approximately 100 nucleotides, both of which could be unstable sequences in bacteria. This work strengthens the genetic power to detect the role of DAT in substance abuse and other neuropsychiatric disorders. Studies of DAT knockout transgenic mice completed during this FY reveal interesting differences in cocaine reward and locomotion.